Severe combined immunodeficiency (SCID) is a group of severe disease which affect immune system. Infants with SCID are healthy at birth but die of recurrent severe infection in infancy unless adequate therapy is provided. Unfortunately, most infants with SCID are not identified in the pre-infection period: the diagnosis is usually hypothesized when a severe infection occurs. At that time, however, even though a correct therapeutic intervention is started, damages due to the severe infection (such meningitis, encephalitis, severe pneumonia) can already be present and permanent sequelae can be an important burden both for the patients and the family, and society.
SCID due to a defect of adenosine deaminase (ADA) or purine-nucleoside phosphorilase (PNP) is an inherited disorder of purine metabolism. Genetic deficiency of the purine salvage enzyme ADA results in varying degrees of immunodeficiency, ranging from neonatal onset severe combined immunodeficiency to late onset immunodeficiency which can determine severe compromise of lung function in adolescents or adults.
In its typical form, the absence of the enzyme ADA allows accumulation of toxic metabolites resulting on one side on severe defect of immune system and, on the other, on permanent damage of other organs and systems such as brain or liver. In these cases SCID-ADA is fatal within the first months of life if untreated and is associate with severe sequelae is treated late.
Late onset ADA-SCID has also been described. In these cases the patients experience severe recurrent infections and chronic lung disease during infancy or adolescence. Very similar are the clinical consequences of PNP defect.
In both cases hematopoietic stem cell transplant is curative, but dependent on a good donor match. Enzyme replacement therapy is available and determines the elimination of toxic metabolites and a good reconstitution of the immune system. Gene therapy is also an option for patients. In any case, whichever therapy is chosen, it should be started as soon as possible after birth in order to obtain good therapeutic effect. Therefore diagnostics methods which allow to make a sure diagnosis in the first days of life are extremely important.
Early diagnosis of ADA-deficiency is necessary because opportune therapies (stem-cell transplantation, enzyme replacement therapy) can be curative while the disease is rapidly fatal if not treated. Diagnosis can be made searching for ADA enzyme activity or for accumulation of metabolites due to ADA deficiency.
Evaluating ADA activity is complex and sometimes can give misleading results: actually a severe defect in ADA activity can be found in subject with an absolutely normal immune function, because variable residual ADA activity expressed in cells different from immune cells can be sufficient to maintain correct immune function. For this reason dosage of metabolites is absolutely mandatory to achieve the diagnosis of immunodeficiency due to ADA or PNP deficiency. Moreover dosage of metabolites allows monitoring the reduction of their toxic activity after starting enzyme replacement therapy.
Measurement of purine and pyrimidine metabolites presents complex problems for separations. Different methods for measurement are used in clinical practice, ranging from HPLC to thin-layer chromatography. Other methods include capillary electrophoresis and even reverse-phase HPLC with electrospray ionisation tandem mass spectrometry.
However all these methods are applied on urine samples and are used when a clinical suspicion of immunodeficiency has already been formulated because of the onset of severe infections. This is a serious drawback of the methods, since affected infants should be diagnosed before onset of infections to maximize opportunity of life-saving treatment. Family history can help in performing and early diagnosis but data obtained in the USA show that only 18% affected patients have a positive family history. The number is probably even lower in Italy where most families have only one child.
The use of mass spectrometry (MS) in clinical laboratories is very much increased on the outset of the 21th century. This development is obviously due to great advances in mass spectrometry applications in the last fifteen years. Mass spectrometry permits a very rapid measurement of different metabolites in different biological specimens using filter paper spots or directly in different biological fluids. Because of its high sensitivity, this technique can be used for qualitative and quantitative analysis of many analytes such as purines and pyrimidines, amino acids and acylcarnitines, homocysteine, orotic acid, succinylacetone etc., with appropriate internal standards.
MS is extensively used for analysis of metabolites from dried blood spots taken at birth (Guthrie-cards) but among the detected metabolites those due to ADA deficiency are not detected because the methods of extraction are not effective. The classical method commonly used for expanded newborn screening is performed by using a C1-3 linear or branched chain monoalcohol (preferably methanol) (Millington D S, et al. J Inherit Metab Dis. 1990; 13(3):321-4; Donald H. et al. Clin. Chem., November 2003; 49: 1797-1817; la Marca G, et al. Rapid Commun Mass Spectrom. 2003; 17(23):2688-92).
Aim of the present disclosure is to provide an analytical method that could allow also the determination of purine and pyrimidine metabolites (including in particular ADA or PNP deficiency metabolites) along with the determination of other metabolites that are commonly determined for metabolites screening, especially those screening performed on dried blood spot taken at birth.